Ethanol (alcohol)-mediated cell and tissue damage is partly caused by increased oxidative and nitrative stress. The majority of reactive oxygen and nitrogen species (ROS/RNS) in alcohol-exposed cells/tissues is being produced through direct inhibition of the mitochondrial respiratory chain and induction/activation of ethanol-inducible cytochrome P450 2E1 (CYP2E1), inducible nitric oxide synthase (iNOS), NADPH-oxidase, xanthine oxidase, etc. Combination of activated CYP2E1, a pro-oxidant enzyme, and suppressed mitochondrial aldehyde dehydrogenase (ALDH2), an anti-oxidant defense enzyme responsible for removal of toxic acetaldehyde and lipid peroxides, leads to increased nitroxidative stress, which not only elevates the amounts of acetaldehyde and lipid peroxides but also contributes to post-translational modification (PTM) of cellular proteins upon exposure to alcohol (ethanol) or other potentially toxic agents. In addition to our effort to characterize oxidatively-modified proteins, we have also established sensitive methods to identify other forms of PTM such as nitration and phosphorylation of various proteins to investigate the roles of protein modifications in promoting mitochondrial dysfunction and acute liver injury. For studying the functional role of protein nitration, we used a mouse model of acetaminophen (APAP)-induced liver injury because of the well-established role of nitration in liver injury. In fact, protein nitration is a contributing factor in alcohol- or APAP-induced liver injury since pretreatment with an iNOS inhibitor such as 1400W prevented tissue injury and that iNOS-null mice were quite resistant to these types of liver injury. However, it was largely unknown which cellular (including mitochondrial) proteins are nitrated and how the functions of nitrated proteins are altered and how they contribute to alcohol- or APAP-mediated mitochondrial dysfunction and hepatotoxicity. By using wild-type (WT) and Cyp2e1-null mice, we recently showed that CYP2E1 is involved in promoting protein nitration and liver injury (Abdelmegeed et al., 2010). Based on our results of the numerous spots of nitrated proteins displayed on 2-D gels, we hypothesized that many more proteins could be nitrated and that nitrated proteins then contribute to liver injury caused by a single dose (350 mg/kg, ip) of APAP, a CYP2E1 substrate. During FY2013, we showed that many cytosolic and mitochondrial proteins were rapidly nitrated at 1 or 2 h following exposure to APAP (4-hydroxyacetanilide) while 3-hydroxyacetanilide (AMAP), a non-toxic analog of APAP, did not promote protein nitration and liver injury. To further study the role of nitrated proteins in liver injury, we purified nitrated proteins via antibody-based affinity columns from mouse livers exposed to APAP for 2 h, where liver damage was minimal. Mass-spectral analysis revealed that more than 30 cytosolic and 65 mitochondrial proteins involved in anti-oxidant defense, energy supply, amino acid and fat metabolic pathways were nitrated by APAP exposure. The enzyme activities of the five selected nitrated proteins including ALDH2, ATP synthase, and 3-ketoacyl-CoA thiolase were suppressed in APAP-exposed mice but restored by co-treatment with an antioxidant N-acetylcysteine, which also prevented protein nitration and liver injury. These results established the causal role of protein nitration in APAP-induced mitochondrial dysfunction and liver injury. We believe that our approach can be used to characterize nitrated proteins in other tissues or disease states. We previously reported the critical role of c-Jun N-terminal protein kinase (JNK) in promoting cell death by phosphorylating critical proteins including pro-apoptotic Bax and mitochondrial ALDH2. To further investigate the roles of JNK and its target proteins in regulating mitochondrial function and cell/tissue damage, we have also characterized JNK-mediated phosphorylation of many mitochondrial proteins. For selective activation of JNK without activating other mitogen-activated kinases, we chose a model of acute liver injury caused by a single dose (50 mg/kg, ip) of carbon tetrachloride (CCL4), another substrate of CYP2E1. We observed that JNK, activated within 30 min, translocated to mitochondria and that many mitochondrial proteins were rapidly phosphorylated between 1 and 8 h after CCL4 exposure where liver injury was minimal. However, these events were not observed in the corresponding Cyp2e1-null mice. To further study their functional roles, we purified phosphorylated mitochondrial proteins from WT mouse livers exposed to CCL4 for 2 h by using metal-affinity columns. Mass-spectral analysis of purified phospho-proteins revealed that more than 100 mitochondrial proteins were phosphorylated by activated p-JNK. These proteins include pyruvate dehydrogenase, ATP synthase, ALDH2, etc involved in energy supply and cellular defense, respectively. The activities of some of these phospho-proteins were markedly suppressed in CCL4-exposed mice but significantly restored in CCL4-exposed mice pretreated with a selective JNK inhibitor SU-3327, which blocked the JNK-mediated protein phosphorylation and liver injury. These novel results provide direct evidence for critical roles of JNK and protein phosphorylation in promoting CCL4-mediated mitochondrial dysfunction and acute liver injury. We have also investigated the role of CYP2E1 in high-fat diet (HFD)-induced nonalcoholic steatohepatitis (NASH) in age- and gender-matched WT and Cyp2e1-null mice. WT mice fed HFD (60% energy derived from fat compared to a low fat diet with 10% fat-derived energy for 10 weeks in a 2 x 2 design) exhibited inflammatory liver injury with increased levels of insulin resistance, nitroxidative stress markers and protein modifications while these characteristics were absent or markedly attenuated in Cyp2e1-null mice. These results demonstrated an important role of CYP2E1 in promoting inflammatory NASH progressed from benign nonalcoholic fatty liver disease (NAFLD). Our results are consistent with the established role of CYP2E1in enhancing alcoholic fatty liver disease (AFLD) and acute tissue injury caused by many hepatotoxic agents. Furthermore, we collaborated with Dr. Ali Keshavarzian at Rush Medical Center in Chicago to investigate the role of CYP2E1 in binge alcohol-mediated gut leakiness, endotoxemia, and advance inflammatory liver disease. Binge alcohol administration (6 g/kg oral gavage for 3 times at 12-h intervals) increased the levels of serum endotoxin, hepatic enterobacterial contents, and hepatic fat accumulation with inflammatory foci in WT mice but not in the corresponding Cyp2e1-null mice. The increased fat accumulation, serum endotoxin, and hepatic contents of enterobacteria were significantly reversed when ethanol-exposed WT mice were co-treated with a specific inhibitor of CYP2E1 chlormethiazole (CMZ) or an antioxidant N-acetylcysteine (NAC). Binge alcohol markedly elevated the levels of nitroxidative marker proteins CYP2E1 and iNOS in intestinal epithelial cells from WT mice. In contrast, these changes were not observed in the ethanol-exposed Cyp2e1-null mice or ethanol-exposed WT mice pretreated with CMZ and NAC, suggesting a critical role of CYP2E1 in gut leakiness and endotoxemia following binge alcohol exposure. The role of PTMs of various tight junction proteins in binge alcohol-mediated gut leakiness, endotoxemia and advanced tissue injury is being investigated. Based on the establishment of AFLD and NAFLD/NASH in our mouse strains, we plan to perform translational research by evaluating the beneficial effects of various anti-oxidants including docosahexaenoic acid against AFLD and NAFLD/NASH in Ppara-null or Cyp2e1-null mice compared to the WT mice.